1. Field of the Invention
The invention relates to a confocal microscope apparatus which excites a specimen which has been marked with a fluorescent dye or fluorescent protein using the excitation wavelength, and detects fluorescence emitted from the specimen.
2. Description of the Related Art
A scanning laser microscope has been proposed, which includes a first optical scanning system for obtaining a scan image of a sample and a second optical scanning system for causing a particular phenomenon in specific areas on the sample surface (refer to Jpn. Pat. Appin. KOKAI Publication No. 2000-275529, the entire contents of which are incorporated herein by reference). In this laser scanning microscope, a specific area on the sample surface is irradiated using a laser light source and an optical path of the first optical scanning system, thus stimulating the sample or a chemical substance injected into the sample. A specific area on the sample surface which is different from the above-mentioned area is excited using a laser light source and an optical path of the second optical scanning system, and the fluorescence is detected, and imaging is carried out. In the specification, unless stated otherwise, an optical scanning system for obtaining images of a sample is called a “first optical scanning system” and an optical scanning system for causing a particular phenomenon in specific areas of a sample is called a “second optical scanning system”.
Generally, in the confocal microscope, the focal point on the sample surface and the conjugated focal point thereof are provided before the detection device, and a pinhole is provided therein. Thereby, the resolution of the sample along the depth direction is 1.22 λ/NA, and a smaller confocal effect is being utilized than when a regular microscope is used for observation. There is resolution as a result of this confocal effect, and thus a sharp cross sectional image (that is, an image to obtain a thin slice image along depth direction) can be obtained for the sample which is being scanned.
When the image is taken at a high speed or when a dark sample is being used, the confocal effect is weakened by opening the pinhole (that is, enlarging a diameter of the pinhole), and the image is made bright by lowering the resolution of the fluorescence.
Thus the confocal microscope has the pinhole and decreases the resolution, and thus depth-direction information can be obtained. However, since the focal depth of the sample is determined by the flux diameter of the coherent light which is irradiated on the objective lens, it is impossible to change the focal depth at the pinhole.
Meanwhile, Koehler illumination is often used as the lighting to the sample by the microscope. This Koehler illumination along the thickness direction of the cross section of the sample causes almost uniform excitation.
In the conventional confocal microscope described above, when the apparatus is realized by using 2 laser scanning paths and one objective lens, the excitation light intensity distribution along the depth direction on the sample surface of the laser beam for sample stimulation and the laser beam for obtaining images are almost the same since only wavelength differences are generated.